Synthesis, Radiolabeling, and Biological Evaluation of (R)- and (S)-2

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Synthesis, radiolabeling, and biological evaluation of (R)- and (S)-2amino-5-[ F]fluoro-2-methylpentanoic acid ((R)-, (S)-[ F]FAMPe) as potential positron emission tomography tracers for brain tumors 18

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Ahlem Bouhlel, Dong Zhou, Aixiao Li, Liya Yuan, Keith Rich, and Jon McConathy J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm502023y • Publication Date (Web): 06 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Synthesis, radiolabeling, and biological evaluation of (R)and (S)-2-amino-5-[18F]fluoro-2-methylpentanoic acid ((R)-, (S)-[18F]FAMPe) as potential positron emission tomography tracers for brain tumors Bouhlel, Ahlem1; Zhou, Dong1; Li, Aixiao1; Yuan, Liya2; Rich, Keith2; McConathy, Jonathan1*. 1

2

Department of Radiology, Washington University in Saint Louis, School of Medicine, Missouri

Department of Neurosurgery, Washington University in Saint Louis, School of Medicine, Missouri.

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Abstract A novel

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F-labeled α,α-disubstituted amino acid-based tracer, 2-amino-5-[18F]fluoro-2-

methylpentanoic acid ([18F]FAMPe) has been developed for brain tumor imaging with a longer alkyl side chain than previously reported compounds to increase brain availability via system L amino acid transport. Both enantiomers of [18F]FAMPe were obtained in good radiochemical yield (24-52% n= 8) and high radiochemical purity (> 99%). In vitro uptake assays in mouse DBT gliomas cells revealed that (S)-[18F]FAMPe enters cells partly via sodium-independent system L transporters and also via other nonsystem A transport systems including transporters that recognize glutamine. Biodistribution and small animal PET/CT studies in the mouse DBT model of glioblastoma showed that both (R)- and (S)[18F]FAMPe have good tumor imaging properties with the (S)-enantiomer providing higher tumor uptake and tumor to brain ratios. Comparison of the SUVs showed that (S)-[18F]FAMPe had higher tumor to brain ratios compared to (S)-[18F]FET, a well-established system L substrate.

Introduction Brain tumor imaging with the most widely used positron emission tomography (PET) tracer for oncology, the glucose analogue 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), is often difficult to interpret due to the high physiologic uptake of [18F]FDG in normal brain and variable [18F]FDG uptake in regions showing post-treatment effects such as radiation necrosis.1-3 In contrast to [18F]FDG, radiolabeled amino acid tracers targeting system L amino acid transporters have superior brain tumor imaging properties due to upregulation of amino acid transporters in gliomas and lower uptake in normal brain.1, 4 These tracers complement anatomic imaging with magnetic resonance imaging (MRI) for definition of tumor margins, monitoring response to therapy, and distinguishing treatment effects from recurrent tumor. System L preferentially transports amino acids with large, neutral side chains (e.g. L-phenylalanine, L-tyrosine, Lleucine) and has four family members: LAT1, LAT2, LAT3, and LAT4. LAT1 and LAT2 are sodium-


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independent and function by exchanging one intracellular amino acid for one extracellular amino acid whereas LAT3 and LAT4 mediate facilitated diffusion of substrates across membranes. System L transporters are active at the luminal side of the blood-brain barrier (BBB) which allows system L substrates to reach the entire tumor volume even when the BBB is not disrupted. A number of wellestablished PET tracers targeting system L transport have been used for human brain tumor imaging including L-[11C]methionine ([18F]MET), O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) and 6-[18F]fluoro3,4-dihydroxy-L-phenylalanine ([18F]FDOPA).4-12 Certain radiolabeled amino acids have also shown promising imaging properties for other types of tumors such as neuroendocrine tumors with [18F]FDOPA and prostate cancer with anti-1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid (anti-3-[18F]FACBC).1317

A range of amino acid transporter systems and substrates have been shown to be relevant to oncologic imaging, and there has been recent interest in PET tracers targeting glutamine transport and metabolism. The metabolically inert tracer anti-3-[18F]FACBC has been shown to be a substrate for ASCT2, an important glutamine transporter in cancer, and to a lesser extent system L and system A, in human prostate cancer cells.18, 19 Human studies indicate that anti-3-[18F]FACBC has clinical potential for imaging prostate cancer and brain tumors.20, 21 Another strategy for targeting glutamine metabolism in cancer has employed a series of

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F-labeled analogues of glutamine. In particular, 4-[18F]-(2S,4R)-

fluoroglutamine ([18F]-FGln) has been shown to be promising in human studies for monitoring progression of gliomas.22-24Additionally, the

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F-tracer targeting system ASC, 3-(1-[18F]fluoromethyl)-L-

alanine (L-[18F]FMA) was shown to be a potential useful PET tracer in preclinical studies.25 A limitation of the tracers FGln and FMA has been in vivo defluorination. There has also been recent interest in glutamate analogues and other amino acids bearing carboxylic acids on their side chains targeting system xC- which is upregulated in many cancers and a marker of tumor oxidative stress.26-28 Finally, lead compounds targeting cationic amino acid transporters including (S)-2-amino-3-[1-(2-[18F]fluoroethyl)-1H-



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[1,2,3]triazol-4-yl]propanoic acid ((S)-[18F]AFETP) and N-2-[18F]fluoroethyl-N-methyl-2-aminoethyltyrosine ([18F]FEMAET) have been recently developed and reported.2, 29, 30

An important limitation of system L transport substrates as imaging agents is the inability of system L transporters to directly concentrate substrates intracellularly. This property leads to relative low tumor to tissue ratios which can reduce sensitivity for lesion detection and lead to washout of activity from tumors over time. Other amino acid transporters including system A transporters can concentrate substrates intracellularly, providing higher and more persistent uptake in tumors. However, the lack of activity of many amino acid transporters at the luminal side of the BBB limits access of their substrates to the enhancing regions of brain tumors. These observations prompted us to develop 18F-labeled amino acid tracers that target both system L and non-system L amino acid transporters in an effort to maintain brain availability through system L transport while enhancing tumor to normal tissue ratios through non-system L transporters. In our previously reports of the synthesis and biological evaluation of three α,αdisubstituted amino acids with varying selectivities for system A and system L transporters: 3-[18F]fluoro2-methyl-2-N-(methylamino)propanoic acid ([18F]MeFAMP), 2-amino-3-[18F]fluoro-2-methylpropanoic acid ([18F]FAMP), and 2-amino-4-[18F]fluoro-2-methylbutanoic acid ([18F]FAMB),31-33 we proposed to develop new 18F-labeled analogues with longer alkyl chain lengths to increase recognition by system L transporters and thereby improve brain availability. Here, we report efficient organic and radiosynthetic routes to obtain the (R)- and the (S)enantiomers of 2-amino-5-[18F]fluoro-2-methylpentanoic acid ([18F]FAMPe), an analogue of [18F]FAMP and [18F]FAMB. These novel tracers were evaluated in the mouse DBT (delayed brain tumor) model of glioblastoma through in vitro uptake assays, biodistribution studies, and small animal PET/CT imaging.2, 34

The tumor and the brain uptake of (R)- and (S)-[18F]8 ((R)- and (S)-[18F]FAMPe) were compared with

the established system L PET tracer, (S)-[18F]FET, and the system A PET tracer (R)-[18F]MeFAMP through small animal PET studies. (S)-[18F]8 demonstrated very high uptake in this tumor model and


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increased recognition by system L transport compared to (R)-[18F]MeFAMP. However, the longer side chain led to loss of recognition by system A transporters with recognition by other neutral amino acid transporters including glutamine transport systems. A. H2N H

B.

O

O OH

H2N H

Me HN Me

OH 18

F

O OH 18

F

O (S)-[18F]FET

OH F

18

18

H2N Me

OH

18

F [18

[ F]MeFAMP

HO

O

O H2N Me

18 F [18F]FAMB

F]FAMP

OH (S)-[18F]FDOPA

O

C. H2N Me

O OH

18

F

(S)-[18F]FAMPe

H2N Me

OH

18

F

(R)-[18F]FAMPe

Figure 1: Selected 18F-labeled amino acids. (A) Both [18F]FET and [18F]FDPOA have been used for imaging brain tumors in human subjects. (B) [18F]FAMP, [18F]FAMB, and [18F]MeFAMP are α,α-disubstituted amino acids; (R)[18F]MeFAMP is an established substrate for system A transport. (C) (S)- and (R)-[18F]FAMPe are novel α,αdisubstituted amino acids evaluated in this publication in the DBT glioma model.

Results and Discussion 1. Chemistry. The racemic non-radioactive forms of the amino acid 2-amino-5-fluoro-2-methylpentanoic acid ((R,S)-8 ) were prepared in a simple eight step synthesis as shown in Scheme 1. From commercially available L-alanine tert-butyl ester hydrochloride and benzophenone imine, we synthesized in very good yield (94%) and without further purification our first intermediate which is the ester imine derivative of alanine, tert-butyl 2-((diphenylmethylene)amino)propanoate ((S)-1). In a second step, we conducted a non-stereoselective alkylation with allyl iodide at the α-carbon of the amino acid derivative which 5 ACS Paragon Plus Environment


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provided tert-butyl (R,S)-2-((diphenylmethylene)amino)-2-methylpent-4-enoate ((R,S)-2), also in very good yield (90%). After the simple and rapid hydrolysis of the imine function of (R,S)-2 with hydroxylamine hydrochloride at room temperature, we obtained the amino acid ester tert-butyl (R,S)-2amino-2-methylpent-4-enoate ((R,S)-3). The following step protected this amine in presence of di-tertbutyl dicarbonate which gave tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-2-methylpent-4-enoate ((R,S)-4) in very good yield (92%). Then, an hydroboration of the alkene followed by hydrolysis in presence of 30% hydrogen peroxide provided the desired terminal alcohol intermediate, tert-butyl (R,S)2-((tert-butoxycarbonyl)amino)-5-hydroxy-2-methylpentanoate ((R,S)-5) in a good yield (62%). The alcohol was converted into a leaving group through tosylation to provide the racemic labeling precursor tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-2-methyl-5-(tosyloxy)pentanoate ((R,S)-6) in moderate yield (40%).

To perform the nucleophilic substitution of the tosylate group with non-radioactive fluoride, we tried several different conditions. Our first attempt consisted of treating the tosylate compound (R,S)-6 with an excess of tetrabutylammonium fluoride in acetonitrile at room temperature. The reaction consumed the starting material to provide a single major product in 86%, but instead of the desired product, an intramolecular cyclization occurred to form di-tert-butyl (R,S)-2-methylpyrrolidine-1,2dicarboxylate ((R,S)-9) as shown in Scheme 2. This reaction gave only trace quantities of the desired fluoro product tert-butyl (R,S)-2-((tert-butoxycarbonyl)amino)-5-fluoro-2-methylpentanoate ((R,S)-7). Because a similar reaction proceeded without this difficulty in the synthesis of FAMB (one carbon shorter side chain),32 the favorable kinetics of the intramolecular formation of a five ring cycle is the likely cause in this case. Attempts to form the di-Boc protected nitrogen compound as has been reported in the synthesis of fluoroleucine derivatives were not successful.35 We thus followed a different protocol in which the authors used tert-amyl alcohol (2-methylbutan-2-ol) as solvent for nucleophilic fluorination reactions.36 The authors suggested that the tert-amyl alcohol solvent system might have four beneficial influences on the mechanism of this fluorination process: (i) the hydrogen bond between the tert-amyl 6 ACS Paragon Plus Environment

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alcohol solvent and the fluoride may reduce the strength of the CsF ionic bond, (ii) the solvation of fluoride will be limited and thus fluoride will be a good nucleophile due to the bulky tert-amyl alcohol, (iii) the hydrogen bond between the tert-amyl alcohol solvent and the oxygen atoms of a sulfonate leaving group may enhance the leaving group reactivity, and (iv) the protic environment of the reaction medium, as well as hydrogen bonding between the tert-amyl alcohol and reactive heteroatoms in the substrate, may reduce side reactions such as intramolecular cyclization. By using three equivalents of cesium fluoride (CsF) in tert-amyl alcohol (2-methylbutan-2-ol) at 80 °C, we obtained the desired fluoro compound (R,S)7 and also the cyclic compound (R,S)-9 after 1 h, but approximately 15% of starting material (R,S)-6 remained. By increasing the amount of CsF used to five equivalents and the temperature to 100 °C without reaching reflux, the reaction was completed in 1 h, and the fluoro compound (R,S)-7 was obtained in 34% yield. We also conducted the reaction in the presence of ten equivalents of CsF but the same yield was obtained. Thus, Kim’s methodology allowed us to obtain the desired product (R,S)-7 in acceptable yields. The final step consisted of the deprotection of (R,S)-7. The deprotection was achieved with 4 M hydrochloric acid at 60 °C which provided the racemic non-radioactive amino acid (R,S)-8 as the hydrochloride salt in good yield (78%) and high purity. Scheme 1: Multistep synthesis of the racemic non-radioactive amino acid (R,S)-8.



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Reagents and conditions: a) diphenylmethanimine, DCM, rt, 2 h; b) LDA, 3-iodoprop-1-ene, THF, -78 °C then rt for 18 h; c) NH2OH, MeOH, rt, 1 h; d) Boc2O, THF, rt, 18 h; e) BH3.THF then NaOH 1 M, H2O2 30%, 0 °C then rt for 18 h; f) TsCl, NaOtBu, DCM, 0 °C then rt for 18 h; g) CsF, 2-methylbutan-2-ol, 100 °C, 1h; h) HCl 4 M, 60 °C, 90 min.

Scheme 2: Reaction conditions (equivalents of fluoride, solvent and temperature) were optimized to minimize the cyclized compound (R,S)-9.

To obtain the enantiomerically pure radiolabeling precursors, tosylate precursors (S)-6 and (R)-6 were prepared in four steps started with the commercially available starting materials (S)-(-)-α-allylalanine and


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(R)-(+)-α-allylalanine respectively (see Scheme 3). During the first step, (S)-α-allylalanine and (R)-αallylalanine were treated with di-tert-butyl dicarbonate to give the carbamates (R)- and (S)-2-((tertbutoxycarbonyl)amino)-2-methylpent-4-enoic acid ((S)-10 and (R)-10), respectively, which were used without purification in the next reaction. The second step consisted of the protection of the carboxylic acid function of (S)-10 and (R)-10 by using N,N-dimethylformamide di-tert-butyl acetal; compounds (S)-4 and (R)-4 were thus obtained. Then, as previously described for the racemic mixture (R,S)-4, the alkene on the alkyl chain was converted into a terminal alcohol which provided products (S)-5 and (R)-5. In the last step, these compounds were converted into tosylates to provide the radiolabeling precursors (S)-6 and (R)-6. Each enantiomerically pure radiolabeling precursor was obtained in similarly good yield.

Scheme 3: Synthesis of the tosylate precursors (S)-6 and (R)-6 for radiolabeling.

Reagents and conditions: a) Boc2O, MeOH/NEt3/NaOH 1 M for 18 h; b) N,N-dimethylformamide di-tert-butyl acetal, anhyd toluene for 2 h at 90 °C then 18 h at rt; c) BH3.THF then NaOH 1 M, H2O2 30%, 0 °C then rt for 18 h; f) TsCl, NaOtBu, DCM, 0 °C then rt for 18 h.

2. Radiochemistry. The radiosynthesis was conducted in two steps (see Scheme 4). The first step, nucleophilic [18F]fluoride substitution, was successfully performed in tert-amyl alcohol (2-methylbutan-2-ol) at 96-105 °C to afford the intermediates (S)-[18F]7 and (R)-[18F]7 from the appropriate enantiomerically pure precursor. Using solid phase extraction with a C18 cartridge, the 18F-labeled intermediate was retained while the unreacted 9 ACS Paragon Plus Environment


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[18F]fluoride was removed. Then, the

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F-labeled intermediate was eluted from the cartridge with

acetonitrile and purified by reverse-phase high performance liquid chromatography (HPLC). The collected fractions from HPLC containing the

18

F-labeled intermediate were combined, diluted with

water, and passed through a light hydrophilic-lipophilic-balanced (HLB) cartridge which was rinsed two more times with water in order to remove residual acetonitrile. The intermediates, (S)-[18F]7 or (R)-[18F]7, were then eluted with a small amount of ethanol. In a second step, a quantitative deprotection was achieved with aqueous hydrochloric acid and followed by elution through a cartridge allowing the neutralization of highly acidic sample to provide the final product (S)-[18F]8 or (R)-[18F]8 which were obtained in 24% to 52% (n= 8) decay corrected yields respectively, at pH= 6 and over 99% radiochemical purity determined by radiometric thin-layer chromatography and analytic HPLC (see Figure 2). In a representative synthesis, a total of 9.9 mCi of (R)-[18F]8 at end of synthesis (EOS) was obtained from 40.8 mCi of [18F]fluoride after the drying process in a synthesis time of approximately 120 min using 5 mg of the tosylate precursor (R)-6. The specific activity was > 1 Ci/µmol at the end of synthesis. Sterile water was used for formulation of the final product for cell uptake assays to avoid the presence of sodium ions in the sodium-free uptake conditions. For the animal studies, the solution was passed through a 0.22 µm nylon filter, and then adjusted to a concentration of 0.9% sodium chloride. The stability of (S)-[18F]8 and (R)-[18F]8 up to 6 h after formulation was confirmed by analytical HPLC.

Scheme 4: Radiosynthesis of (S)-[18F]8.


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Figure 2: Analytical chiral HPLC co-injections of a mixture of the non-radioactive racemic product (R,S)-8 (seen with UV detection) and (S)-[18F]8 (seen with radiometric detection).

3. Cell uptake assays. Amino acid uptake assays were performed using mouse DBT glioma cells in the presence and absence of two well-described inhibitors of amino acid transport, N-methyl α-aminoisobutyric acid (MeAIB) and 2-amino-bicyclo[2.2.1]-heptane-2-carboxylic acid (BCH), which were used as inhibitors for system A and system L, respectively. These two commonly used inhibitors allowed us to evaluate the contribution of these amino acid transport systems to the cellular uptake of (S)- and (R)-[18F]8. The results of the in vitro cell uptake assays are shown in Figure 3. The two enantiomers were compared to (R)[18F]MeFAMP, a good substrate for system A amino transport, and (S)-[18F]FET, a well-characterized system L transport substrate. The uptake assays were performed with a brief (30 or 60 seconds) uptake time to evaluate the initial influx of (S)- or (R)-[18F]8 and minimize the potential for efflux which occurs with many amino acid transporters including system L and ASC. An additional set of inhibition



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experiments shown in Figure 4 were performed using varying concentrations of L-glutamine (Gln) to establish the role of glutamine transporters in the uptake of (S)- and (R)-[18F]8. Certain amino acids transporters including system A and system ASC require co-transport of sodium ions to function while others including system L function in sodium-free conditions. Thus, we compared uptake in the presence of sodium ions and under sodium-free conditions using the cation choline as a substitute for sodium ions. In presence of MeAIB, there was no significant inhibition uptake of (S)-[18F]8, indicating that system A transport does not play a substantial role in the in vitro uptake of this tracers by DBT cells. A minor component of inhibition of (R)-[18F]8 was observed with MeAIB (83 ± 17% uptake relative to control, p < 0.05). These results are similar to the ones obtained with the structurally similar compound, [18F]FMA, developed by Wang et al., as uptake of this tracer by 9L glioma cells was not inhibited by MeAIB.25 Substitution of choline for sodium ions resulted in partial inhibition of uptake of both enantiomers with 71 ± 17 % uptake relative to control for (S)-[18F]8 and 70 ± 11% for (R)-[18F]8. These results indicate that both enantiomers enter DBT cells partly via sodiumdependent amino acid transport, but not via the sodium-dependent system A, as shown by the lack of uptake inhibition by MeAIB. In the presence of MeAIB, the uptake of the system A substrate (R)[18F]MeFAMP was 19 ± 9% relative to the sodium control which is consistent with prior reports that (R)[18F]MeFAMP is a good substrate for system A in a range of rodent and human cancer cell lines.31, 33, 37 Similarly, the uptake of (R)-[18F]MeFAMP was reduced to 21 ± 19% relative to the sodium control in the sodium-free choline condition, consistent with sodium-dependence of system A transport and demonstrating that (R)-[18F]MeFAMP enter DBT cells mainly via the system A amino acid transport. As expected, uptake of the system L transport substrate (S)-[18F]FET by DBT cells was not significantly inhibited by MeAIB, inhibitor of the system A amino acid transporter, or by sodium-free conditions as system L is sodium-independent. BCH is commonly used as a competitive antagonist of the sodium-independent L type transport system, but also competitively inhibits amino acid uptake via the sodium-dependent B0,+ and B0 transport system. In the presence of sodium, BCH reduced the uptake of (S)-[18F]8 to 59 ± 15% relative to control 12 ACS Paragon Plus Environment

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and reduced the uptake of (R)-[18F]8 to 47 ± 12% relative to control. In the absence of sodium, the addition of BCH reduced the uptake of (S)-[18F]8 and (R)-[18F]8 to 72 ± 11% (p = 0.017) and 46 ± 4% (p < 0.001) respectively, relative to the choline control condition. These results indicate a component of system L transport of both compounds. In the case of (R)-[18F]MeFAMP, no significant inhibition was observed in the presence of sodium BCH relative to control. In contrast, almost all of the uptake of (S)[18F]FET was inhibited by BCH in the presence and absence of sodium (p < 0.01), consistent with prior studies showing (S)-[18F]FET is substrate for system L amino acid transport.9 Equal concentrations of the amino acids alanine, serine, and cysteine (ASC) were also used for uptake inhibition as these amino acids are competitive inhibitors for many neutral amino acid transporters including system A, system ASC, and to a lesser extent system L. In the sodium ASC condition, the uptake of (S)-[18F]8 and (R)-[18F]8 were reduced to 52 ± 14% and 41 ± 4%, respectively, relative to the sodium control (p < 0.001 for both). These results indicate that (R)- and (S)-[18F]8 undergo uptake by non-system A neutral amino acid transport in addition to a component of system L transport. In the case of (R)-[18F]MeFAMP, the ASC condition reduced the uptake of (R)-[18F]MeFAMP to 22 ± 9% relative to the sodium control (p < 0.001); the uptake of (S)-[18F]FET was reduced by approximately 40% but did not reach statistical significance. Because glutamine is an important amino acid in cancer metabolism and the ASC condition is expected to inhibit glutamine uptake, additional uptake assays were performed in presence of different concentrations of glutamine (0.1, 1.0 and 10 mM) and ASC (1 mM and 10 mM) as shown in Figure 4. The results demonstrated that (S)-[18F]8 uptake was more sensitive to glutamine and ASC inhibition than (R)-[18F]8. The uptake of (S)-[18F]8 was blocked in a dose-dependent manner by glutamine and ASC; in contrast, significant inhibition of (R)-[18F]8 did not occur in the presence of glutamine and was observed only with the 10 mM ASC condition. At the 1 mM and 10 mM concentration of glutamine, the uptake of (S)-[18F]8 was respectively reduced to 76 ± 21% (p < 0.05) and 66 ± 9% (p < 0.001) relative to the control value. Similarly, at the 1 mM and 10 mM concentrations of ASC, the uptake of (S)-[18F]8 uptake was respectively reduced to 61 ± 16% (p < 0.01) and 38 ± 9% relative to the control value. In contrast, only 13 ACS Paragon Plus Environment


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the 10 mM concentration of ASC caused a significant reduction of (R)-[18F]8 uptake (49 ± 9% relative to control, p < 0.001). These data suggest that (S)-[18F]8 uptake by DBT cells is partly mediated by amino acid transporters that recognize glutamine.

Figure 3: In vitro uptake of (S)- and (R)-[18F]8, (R)-[18F]MeFAMP and (S)-[18F]FET in DBT glioma cells in presence and absence of competitive inhibitors of amino acid transport. The uptake data are normalized based on the amount of activity added to each well and the total amount of protein in each well. The data are expressed as percentage uptake relative to the sodium control condition, and the values for each condition are noted in the appropriate bars. The number of replicates per condition were 7 or 8 for (S)- and (R)-[18F]8 and 3 or 4 for (R)[18F]MeFAMP and (S)-[18F]FET. Na control and Cho control contain 10 mM sucrose to provide an osmolarity consistent with the inhibitory conditions. Na= assay buffer containing sodium ions; Cho= assay buffer containing choline ions; MeAIB= 10 mM N-methyl α-aminoisobutyric acid (system A inhibitor); BCH= 10 mM 2-aminobicyclo[2.2.1]-heptane-2-carboxylic acid (system L inhibitor); ASC= 3.3 mM each of L-Ala, L-Ser, L-Cys. p-values represent comparisons of uptake in the presence of inhibitor to control uptake for each radiotracer (1-way ANOVA) with Dunnett’s multiple comparison post-tests . The choline sucrose control condition was compared to the choline BCH condition using a 2-sided t-tests. * = p < 0.05, ** = p = 0.017, † = p

Synthesis, Radiolabeling, and Biological Evaluation of (R)- and (S)-2

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